Cloning and characterization of lin genes responsible for the degradation of Hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90

Abstract

Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta. While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S. paucimobilis B90. The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation. The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S. paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S. paucimobilis UT26. No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.

FIG. 1.

Degradation of HCH by S. paucimobilis B90 (a) and S. paucimobilis UT26 (b) in SM plus 1% glucose. An initial inoculum of 0.5 ml (10⁸ cells/ml) was added to 50 ml of medium, and simultaneously, each HCH isomer was added separately (5 μg/ml). Samples were withdrawn periodically, extracted with hexane, and analyzed on a gas chromatograph equipped with an electron capture detector. ♦, α-HCH; ▪, β-HCH; •, γ-HCH; □, δ-HCH. The error bars indicate standard deviations.

FIG. 2.

Southern blot hybridization of genomic DNA of S. paucimobilis B90 (a and b) and strain UT26 (b) with an [α-³²P]dATP-labeled truncated fragment of linA. (a) Lane 1, lambda DNA digested with EcoRI and HindIII; lanes 2 to 7, B90 DNA digested with BamHI, BclI, BglII, EcoRI, HindIII, and SalI, respectively; lane 8, amplified linA1 (462 bp) as a positive control. (b) Lane 1, amplified linA1 (462 bp) as a positive control, lanes 2 to 5, genomic DNA digested with HindIII (UT26), HindIII (B90), BclI (UT26), and BclI (B90), respectively; lane 6, lambda DNA digested with EcoRI and HindIII.

FIG. 3.

Schematic drawings of the regions from S. paucimobilis UT26 and B90 comprising the linA genes. (a) Reconstructed drawing from the published DNA sequences of linX (accession no. D23722) (20) and linA (accession no. D90355) (6) of S. paucimobilis UT26. (b) reconstructed drawings based on the sequences of linA1 and linX of S. paucimobilis B90. (c) The linA2 region. (d) Trace of Tn610 of M. fortuitum (accession no. X536535) (14a). Similar drawings (bold and dotted lines) indicate regions of DNA sequence homology between different genes or regions.

FIG. 4.

Detected instability of lin genes in strain UT26. Shown are products of PCR amplification with primers for linA, -B, -C, -D, and -X and the Tn610-like sequence on total DNA of S. paucimobilis B90 and UT26 isolated from individual clones. Lanes: 1 and 2, Tn610 detection in strain B90 (lane 1) and UT26 (lane 2); 3 and 4, linX in UT26 (lane 3) and B90 (lane 4); 5 and 6, linD in UT26 (lane 5) and B90 (lane 6); 7 and 8, linC in UT26 (lane7) and B90 (lane 8); 9 and 10, linB in UT26 (lane 9) and B90 (lane 10); 11 and 12, linA in UT26 (lane 11) and B90 (lane 12); 13, lambda DNA digested with EcoRI and HindIII.

FIG. 5.

Degradation of γ-HCH by E. coli BL21(pLINEA1) or BL21(pGEX-5X-3). (a) Conversion of 5 μg of γ-HCH/ml by pLINEA1 and pLINEA2 in E. coli BL21 growing in LB medium. Samples were drawn periodically and analyzed by gas chromatography. ▴, control; ♦, pLINEA1; ▪, pLINEA2. (b) Degradation of γ-HCH by cell extracts of E. coli BL21(pLINEA1) expressing the linA1 gene. The cell extracts contained 5 mg of protein/ml, to which 5 μg of γ-HCH/ml was added. ▴, control; ♦, pLINEA1; ▪, pLINEA2.

FIG. 6.

Possible evolutionary origin of HCH degradative genes and pathways in S. paucimobilis B90 and S. paucimobilis UT26.